The transcription machinery is the ultimate target of many signal transduction and developmental pathways and regulation of transcription is one of the key steps in control of cell growth, differentiation and development. Defects in transcription and its regulation directly contribute to human illnesses such as cancer, inflammation, heart disease, neurological disorders, and birth defects. The broad long-term objective of this proposal is to determine the mechanism of transcription initiation by eukaryotic RNA polymerase II. Understanding the molecular mechanisms of the transcription process will form the basis for understanding gene control and the action of transcription regulators, many of which modulate the activity of the transcription machinery at various points in the recruitment, initiation, and elongation steps. The specific aims of this work will utilize biochemical, molecular, genetic, and structural methods to examine the mechanism of transcription initiation by S. cerevisiae RNA Pol II. Using biochemical methods we have developed for mapping the structural arrangement of large complexes, in conjunction with a purified transcription system, we will map the structure of the transcription machinery at intermediate stages of the transcription cycle: Preinitiation Complex, Open Complex, initiation site-scanning, initiation, and promoter escape. We will use yeast molecular genetics to test the significance of interactions observed in biochemical assays. Combined, our results will lead to a detailed model for the mechanism of transcription initiation by Pol II and the role of the general transcription factors in this process. PUBLIC HEALTH RELEVANCE: The objective of this research is to understand the mechanism and regulation of transcription, the process of mRNA synthesis. Regulation of transcription is one of the key steps in control of cell growth, differentiation, and development, and defects in transcription directly contribute to many human illnesses. Understanding the mechanism of transcription and its regulation will form the basis for understanding the molecular defects in transcription disorders leading to many types of cancer, as well as heart disease, neurological disorders, and birth defects.